Trigging stepwise-strand displacement amplification lights up numerous G-quadruplex for colorimetric signaling of serum microRNAs

Summary MicroRNAs (miRNAs) play an important biomarker in various biological processes, especially cancer related, yet economic, simple, sensitive and specific methods for miRNA determination are still challenging. In this study, we have developed stepwise-strand displacement amplification (S-SDA)-based colorimetric sensing platform for let-7a miRNA detection in clinical serum samples. Our results demonstrated that the developed S-SDA-based method shows high sensitivity with a detection limit of 63.2 pM and a naked eye detection limit of 0.1 nM. Moreover, the S-SDA amplifier is able to discriminate target miRNAs from their mutants with high accuracy and specificity. With its high sensitivity and selectivity, this method successfully identified healthy individuals from patients with colon cancer by detecting let-7a miRNAs in serum. We believe the colorimetric analysis method will provide a new paradigm for the detection of miRNA with different abundance and show great potential for clinical application in biomedical analysis and early clinical diagnosis.


INTRODUCTION
MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs (ca. 18-24 nt), and as important gene regulators, they are widely involved in a series of biological processes, such as cell differentiation, expression, cellular proliferation, and so on. 1,2 A large number of studies have shown that the abnormal expression of miRNA is linked to the pathogenesis of a variety of human genetic diseases, [3][4][5][6] such as cancer, cardiovascular disease, diabetes, and neurodegenerative diseases. [7][8][9] Therefore, quantitative detection and analysis of miRNAs expression is of great significance for early diagnosis and treatment of related diseases. However, due to the short length, low abundance, and high sequence similarity of family members, it makes difficulties to quantify the expression level of miRNAs.
So far, researchers have disclosed different detection techniques and methods, including PCR, 10 northern blotting, 11 and DNA microarrays. 12 However, these traditional nonamplified methods always need high criteria for instruments, high cost, complicated operation, poor sensitivity, and false-positive results. Therefore, new signal amplification strategies method for analyzing miRNAs has been developed, including hybridization chain reactions, 13 catalytic hairpin assembly, 14 strand displacement amplification (SDA), 15,16 and rolling circle amplification. 17,18 Especially, SDA has become an attractive tool for quantitative detection and analysis of miRNAs expression in the past decade due to its simple operation, isothermal reaction, rapid amplification kinetics, and high sensitivity. However, most of the reported SDA-based methods usually require the labeling of fluorescence, organic dyes, quantum dots, or nanoparticles in these designs, which leads to the increase of the cost and sophisticated processes. In an alternative approach, the visual sensing miRNA has become a compelling emerging research field in recent years due to its easy handling and commercialization. Recently, the G-quadruplex-hemin DNAzyme that is similar to horseradish peroxidase could catalyze the oxidation of 3,3 0 ,5,5 0 -tetrazmethylbenzidine sulfate (TMB) or 2,2 0 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid disodium salt) (ABTS 2À ) by H 2 O 2 19 has been increasingly used for colorimetric, 20 electrochemical, 21 and/or chemiluminescence 22 detection of a variety of target, including DNA, protein, metal ion, and telomerase activity. As compared with protein enzymes, the intrinsic merit of DNAzymes depends on their versatility of design and low cost. However, the SDA-based method always suffers from low signal-to-noise ratio problems due to inevitable background amplification, 23 which limits the realization of detection sensitivity.
Here, we wish to develop a colorimetric-based autonomous polymerization/nicking machinery for miRNA (using let-7a miRNA as a model) detection. By coupling G-quadruplex DNAzyme-based sensing systems with the significant amplification efficiency of SDA, let-7a miRNA could trigger the stepwise-SDA (S-SDA) to form plenty of G-quadruplex, providing an amplified readout signal of the target miRNA and, thus, offering high detection sensitivity. Meanwhile, due to the excellent discriminative ability of S-SDA employed, the high specificity of this method was obtained. In addition, this strategy has been successfully developed for colorimetric analysis of let-7a miRNA in a complex condition and clinical serum samples. With these advantages, this efficient S-SDA-based autonomous machinery holds a significant potential in biochemical studies and clinical diagnosis.

Principle of the proposed strategy
The principle of target let-7a miRNA-induced S-SDA is illustrated in Scheme 1. Briefly, this system mainly consists of only two linear DNA template probe (LDTP1 and LDTP2), DNA polymerase, and nicking endonuclease. The LDTP1 consists of two domains: ① let-7a miRNA recognition region and ② a recognition domain for Nt.BbVCI, which could recognize (5 0 -CCTCAGC-3 0 /3 0 -GGAGTCG-5 0 ) and specifically cleave the upper double-stranded DNA (dsDNA). In the presence of target miRNA, it will specifically bind to LDTP1, and the 3 0 end of miRNA can be regarded as a polymerized primer to initiate polymerization with the help of KF polymerase and dNTPs, which synthesizes a duplex conformation. Then, nicking endonuclease Nt.BbVCI specifically cleaves the upper DNA duplex. Through the repeated extension and cleavage via SDA1 reaction, abundant DNA triggers are displaced. Furthermore, the released DNA triggers are free to bind to LDTP2 to initiate the second extension and cleavage cycle via SDA2 reaction. This S-SDA operation would allow for the production of plenty of DNAzymes that triggers the subsequent oxidation of TMB by H 2 O 2 in the presence of hemin for colorimetric signal transduction. Conversely, the S-SDA reaction cannot be initiated and the hemin/G-quadruplex DNAzyme structures cannot be formed without target miRNA. Due to the high amplification efficiency of SDA1 and SDA2, one target miRNA could be amplified for colorimetric detection with high sensitivity. To verify the feasibility of the proposed method for let-7a miRNA analysis, UV-vis spectra were used. As shown in Figure 1A, in the case of no target miRNA, the G-quadruplex sequence was almost difficult to form and the absorption peak (650 nm) was very low (curve a), indicating that no S-SDA reaction executes and no DNAZyme is produced. Whereas obvious enhanced absorption was observed in the presence of let-7a miRNA (curve b), which is slightly lower than with the use of G-quadruplex sequence only (curve c). These results indicated the occurrence of S-SDA reactions and generation of some hemin/G-quadruplex DNAzyme upon the addition of target miRNA. This UV-vis spectra result was in good agreement with the result of naked eye observation. As shown in Figure 1A inset, a clear green color was observed by the naked eye in the presence of let-7a miRNA, indicating the detection and discrimination process can be seen with the naked eye without the need of any complicated measuring instrument. The designed S-SDA fabrication and recognition process has also been confirmed by 12% non-denaturing PAGE. As shown in Figure 1B

Sensitivity of miRNA assay
Under the optimized experimental conditions ( Figure S1), a series of different concentrations of target miRNA (0-400 nM) were measured to estimate the sensitivity and linear range of the colorimetric method for let-7a miRNA detection. As shown in Figure 2A, it can be observed clearly that the UV-vis absorption intensity increases with the increase of let-7a miRNA concentration from 0 to 400 nM and the solution color changes from colorless to green with the increasing amount of added let-7a miRNA. Notably, in the range from 0 to 400 nM, the absorption peak value at 650 nm shows a linear correlation with 1/4th root of concentration of let-7a miRNA ( Figure 2B). The correlation equation is A = 0.0082C 1/4 + 0.1089 (correlation coefficient was 0.9911), where A and C are the absorption peak value at 650 nm and the concentration of let-7a miRNA (pM), respectively. According to the equation the limit of detection (LOD) = the average absorption value of the blank + three times the standard deviation, LOD was calculated to be 63.2 pM for instrument detection. Such sensitivity is superior or comparable to many other enzyme-assisted amplified sensors (Table S2). Notably, there occurred distinct color change upon addition of 0.1 nM. Therefore, the LOD iScience Article for naked eye detection was estimated to be 0.1 nM. This is attributed to the high efficiency of S-SDA for signal amplification, making it more promising for application in early diagnosis with high sensitivity.

Detection specificity
To assess the selectivity of the developed S-SDA, different miRNAs, including let-7a, miRNA-31, miRNA-199, miRNA-203, miRNA-210, miRNA-141, miRNA-26a, miRNA-145, and random miRNA, are selected as the detection model. As shown in Figure 3, noncomplementary targets as interfering agents (miRNA-31, miRNA-199, miRNA-203, miRNA-210, miRNA-141, miRNA-26a, miRNA-145, and random miRNA) do not cause absorbance signal changes, which was similar to that of the blank, but significant absorbance intensity at 650 nm was observed in the presence of target miRNA (let-7a miRNA). Similarly, the color of the solution shows the same trend. As shown in Figure 3 inset, a remarkably distinguishable color change can also be observed for let-7a miRNA compared with other miRNAs. To further investigate the specificity of the S-SDA-based method, the nonspecific miRNAs of the other members of let-7 family (let-7b, let-7c, let-7d, and let-7i) were tested with the same concentration. As shown in the Figure S2, compared with the let-7a miRNA, the absorbance signal or color changes of the miRNA family members with one, two, or  iScience Article four mismatched nucleotides showed nearly negligible. These results above indicated that our S-SDAbased colorimetric sensing platform has superb selectivity for let-7a miRNA toward other competitive miRNAs.

Detection of let-7a miRNA in real samples
To investigate the practicality of the S-SDA-based colorimetric sensing platform for miRNA detection in real biological samples, let-7a miRNA was added into the commercial fetal bovine serum (FBS) and absorbance measurements were monitored. As shown in Figure 4, there was no statistically significant change in the S/N (approximately 3.4) after their incubation in 0% and 5% FBS. To further test the practical utility of S-SDA-based sensing platform, various concentrations of let-7a miRNA were added in 1000-fold and 100-dilution of human serum samples that obtained from healthy person. The detected results were shown in Table S3 and exhibited good recovery. These results indicate that the developed S-SDA-based colorimetric sensing platform can be successfully applied to complex biological environment.
To further demonstrate the practical application of the S-SDA-based colorimetric sensing platform, serum samples derived from patients with colon cancer and non-cancerous healthy serum sample (healthy person) which obtained from Jiujiang First People's Hospital, and absorbance intensity was monitored ( Figure 5A). In order to simplify the detection, blood samples were centrifuged to obtain the corresponding serum, and then heated at 95 C for 3 min to inactivate interfering nucleic acids-degrading enzymes that might interfere with S-SDA detection. As shown in Figure 5B, the absorbance of patients' sample with colon cancer was significantly higher compared to non-cancerous healthy sample. The obtained data indicated that let-7a miRNAs are overexpressed in cancer samples. What is pleasant is that patients with cancer are easily distinguished from healthy people with the naked eye by using the method (Figure 5B inset). Furthermore, miRNAs were detected blindly in order to demonstrate S-SDA's ability to distinguish target miRNA from nontarget miRNA. Based on their relative absorption intensities, target miRNA (let-7a miRNA) and nontarget miRNA can be distinguished from one another in Table S4. Assay results from the blind test were in agreement with laboratory assistant's pre-made marks. These preliminary results demonstrated that the S-SDA-based colorimetric sensing platform could be useful in clinical settings for colon cancer diagnosis by using clinical serum samples.

DISCUSSION
In summary, we have developed S-SDA-based colorimetric sensing platform for let-7a miRNA detection. Taking advantage of the high amplification efficiency of dual-stage isothermal amplification reaction, highly sensitive visual detection of let-7a miRNA by the naked eye without using any sophisticated instruments was achieved. We have proved that S-SDA-based method exhibited excellent selectivity and was successfully applied to detect let-7a miRNA in a complex matrix samples including the FBS and clinical serum

Limitation of the study
In this study, let-7a miRNA levels were found to be higher in patients with colon cancer than in healthy people; however, the accuracy of this approach needs to be verified in case of the real applications due to the interference of other substances in blood.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: